The present invention relates to a cell culture vessel having excellent cell separatability, a production process thereof, and cells cultured in such a culture vessel.
The recent years have seen remarkable progress of the techniques for culture of cells used for medical purposes, and the cultured cells are actually used for grafting of the skin. There has also been seen progress in cell culturing techniques to be applied to autologous transplantation and heterologous transplantation, which application is not limited to grafting of simple tissues such as skin but extends to transplantation of more complex organs such as cornea, tooth, bone and viscera.
Various vessels such as glass or resin-made Petri dish are used for culture of cells. For instance, the Petri dishes for cell culture made by the following methods have been disclosed. In one method, a collagen solution of a specified concentration is pipetted to a culture vessel for coating such as to form a uniform surface and then dried for 15 minutes to 72 hours. In another method, a collagen solution of a specified concentration is coated on a flexible culture base material such as silicone membrane and polymerized in a 15-42° C. incubator for 20-120 minutes, then the flexible culture substrate is left under UV lamp irradiation for 15 minutes to 72 hours, and after collagen has been dried away, the substrate is again wetted with a phosphate butter solution, then extended 10-40% and fixed (see JP-A-2002-142751, e.g., Example A).
According to the first method, although it is possible to culture a cell on collagen having affinity for the cell, difficulties are involved in separating the cell because of strong adhesion between the cell and the culture vessel. If the cell is separated by mechanical means, physical damage may be given to the cell, and when a chemical treatment with an enzyme such as trypsin is applied on the cell, the membrane protein in the cell surface may be destroyed to reduce the cell fixing rate to the tissue after grafting.
The present inventors previously disclosed, in the below-mentioned patent, a cell culture vessel free of the above problem. In this patent, the cell is cultured on a functional substrate provided with organic polymer-made columnar microprojections capable of shape controlling, whereby the culture solution can be readily placed below the cell and also cell separatability is improved (see JP-A-2004-170935, e.g., Example 5).
According to the second method mentioned above, the problem relating to separation of the cell is solved by lowering adhesion between the cell and the culture vessel by reducing the area of the culture vessel surface where the cell is brought into contact. This method, however, had the problem that the cell would be adsorbed even to the sheet end or the portion of the dish bottom where the functional substrate is absent, causing a drop of cell culturing efficiency. Further, in case the cells cultured on the microprojections exhibit the properties different from those shown when the cells are cultured in an ordinary culture vessel, the cells cultured on the microprojections may be mixed with the cells cultured on a flat portion having no microprojections.
It is therefore an object of the present invention to provide a cell culture vessel which is simple in structure and easy to handle, and is capable of preventing damage to the cells when separated, promoting transport of nutrients and excretion of effete matter, and elevating the culturing efficiency improving effect by the structural features.